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Rap1 promotes cell spreading by localizing Rac guanine nucleotide exchange factors.

Arthur WT, Quilliam LA, Cooper JA - J. Cell Biol. (2004)

Bottom Line: Rap1 is necessary for the accumulation of VAV2 in membrane protrusions at the cell periphery.In addition, if VAV2 is artificially localized to the cell edge with the subcellular targeting domain of Rap1a, it increases cell spreading independently of Rap1.These results lead us to propose that Rap1 promotes cell spreading by localizing a subset of Rac GEFs to sites of active lamellipodia extension.

View Article: PubMed Central - PubMed

Affiliation: Fred Hutchison Cancer Research Center, Seattle, WA 98109, USA. barthur@fhcrc.org

ABSTRACT
The Ras-related GTPase Rap1 stimulates integrin-mediated adhesion and spreading in various mammalian cell types. Here, we demonstrate that Rap1 regulates cell spreading by localizing guanine nucleotide exchange factors (GEFs) that act via the Rho family GTPase Rac1. Rap1a activates Rac1 and requires Rac1 to enhance spreading, whereas Rac1 induces spreading independently of Rap1. Active Rap1a binds to a subset of Rac GEFs, including VAV2 and Tiam1 but not others such as SWAP-70 or COOL-1. Overexpressed VAV2 and Tiam1 specifically require Rap1 to promote spreading, even though Rac1 is activated independently of Rap1. Rap1 is necessary for the accumulation of VAV2 in membrane protrusions at the cell periphery. In addition, if VAV2 is artificially localized to the cell edge with the subcellular targeting domain of Rap1a, it increases cell spreading independently of Rap1. These results lead us to propose that Rap1 promotes cell spreading by localizing a subset of Rac GEFs to sites of active lamellipodia extension.

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Rap1 targets VAV2 to circumferential membrane protrusions. (A) Both active and inactive Rap1a localize to membrane protrusions. HeLa cells were transiently transfected with vectors encoding GFP-63E Rapla (63E) or GFP-17N Rap1a (17N) alone (left) or cotransfected with a HA-SWAP-70–encoding vector (right four panels). Transfected cells were suspended, plated on fibronectin for 1 h, fixed, and labeled with HA antibodies. Note that cells expressing 17N Rap1a do not spread, but when spreading is induced with SWAP-70, then 17N Rap1a is detected at the cell periphery. Arrowheads indicate localization of the GFP-Rap1a variants at the cell edge. (B) VAV2, but not COOL-1, requires Rap1 activity to localize to membrane protrusions. HeLa cells were transiently cotransfected with vectors encoding Myc-VAV2 or Myc-COOL-1 and GFP or GFP-Rap1GAP together with HA-SWAP-70. HA-SWAP-70 was cotransfected with the Rac GEFs and GFP vectors to allow Rap1-independent spreading. Transfected cells were treated as in A, labeled with Myc antibodies, and only well-spread cells were analyzed. Arrowheads indicate localization of the GEFs at the cell edge.
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fig7: Rap1 targets VAV2 to circumferential membrane protrusions. (A) Both active and inactive Rap1a localize to membrane protrusions. HeLa cells were transiently transfected with vectors encoding GFP-63E Rapla (63E) or GFP-17N Rap1a (17N) alone (left) or cotransfected with a HA-SWAP-70–encoding vector (right four panels). Transfected cells were suspended, plated on fibronectin for 1 h, fixed, and labeled with HA antibodies. Note that cells expressing 17N Rap1a do not spread, but when spreading is induced with SWAP-70, then 17N Rap1a is detected at the cell periphery. Arrowheads indicate localization of the GFP-Rap1a variants at the cell edge. (B) VAV2, but not COOL-1, requires Rap1 activity to localize to membrane protrusions. HeLa cells were transiently cotransfected with vectors encoding Myc-VAV2 or Myc-COOL-1 and GFP or GFP-Rap1GAP together with HA-SWAP-70. HA-SWAP-70 was cotransfected with the Rac GEFs and GFP vectors to allow Rap1-independent spreading. Transfected cells were treated as in A, labeled with Myc antibodies, and only well-spread cells were analyzed. Arrowheads indicate localization of the GEFs at the cell edge.

Mentions: As a second test for VAV2 localization, we examined the subcellular distribution of Rap1 and VAV2 in spreading cells using immunofluorescence (Fig. 7). Consistent with a recent paper (Bivona et al., 2004), a fraction of activated 63E Rap1a, but not dominant-negative 17N Rap1a, was found in membrane protrusions around the circumference of transiently transfected cells (Fig. 7 A, left). The absence of 17N Rap1a at the cell periphery conflicted with our pseudopodia fractionation studies and could be a secondary effect due to the lack of membrane protrusions around the circumference of cells in which Rap1 is inhibited. Accordingly, the Rac GEF SWAP-70, which induces cell spreading independently of Rap1, was expressed with 63E or 17N Rap1a. Consistent with the pseudopodia fractionation results (Fig. 6), both 63E Rap1a and 17N Rap1a were enriched in circumferential protrusions in SWAP-70–expressing cells (Fig. 7 A). These data suggest that both active and inactive Rap1 localize to Rac-dependent membrane protrusions. We examined the localization of the Rap1-dependent and -independent Rac GEFs VAV2 and COOL-1 in cells with normal or attenuated Rap1 activity. Overexpressed VAV2 and COOL-1 were both in peripheral membrane ruffles in cells with normal Rap1 activity. However, when Rap1 was inhibited with Rap1GAP, VAV2 but not COOL-1 was displaced from the cell periphery (unpublished data). Because the cells expressing VAV2 and Rap1GAP were poorly spread (Fig. 4), we again used SWAP-70 to induce Rap1-independent spreading (Fig. 7 B). Under these conditions, the cells were well-spread regardless of Rap1 activity, but VAV2 was displaced from circumferential membrane protrusions when Rap1 was inactivated. Together, these experiments suggest that active Rap1 relocalizes Rac GEFs that bind to Rap1, such as VAV2, to the periphery of spreading cells. In contrast, Rac GEFs that do not bind Rap1, such as COOL-1, accumulate in the periphery of spreading cells independently of Rap1 activity.


Rap1 promotes cell spreading by localizing Rac guanine nucleotide exchange factors.

Arthur WT, Quilliam LA, Cooper JA - J. Cell Biol. (2004)

Rap1 targets VAV2 to circumferential membrane protrusions. (A) Both active and inactive Rap1a localize to membrane protrusions. HeLa cells were transiently transfected with vectors encoding GFP-63E Rapla (63E) or GFP-17N Rap1a (17N) alone (left) or cotransfected with a HA-SWAP-70–encoding vector (right four panels). Transfected cells were suspended, plated on fibronectin for 1 h, fixed, and labeled with HA antibodies. Note that cells expressing 17N Rap1a do not spread, but when spreading is induced with SWAP-70, then 17N Rap1a is detected at the cell periphery. Arrowheads indicate localization of the GFP-Rap1a variants at the cell edge. (B) VAV2, but not COOL-1, requires Rap1 activity to localize to membrane protrusions. HeLa cells were transiently cotransfected with vectors encoding Myc-VAV2 or Myc-COOL-1 and GFP or GFP-Rap1GAP together with HA-SWAP-70. HA-SWAP-70 was cotransfected with the Rac GEFs and GFP vectors to allow Rap1-independent spreading. Transfected cells were treated as in A, labeled with Myc antibodies, and only well-spread cells were analyzed. Arrowheads indicate localization of the GEFs at the cell edge.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2172522&req=5

fig7: Rap1 targets VAV2 to circumferential membrane protrusions. (A) Both active and inactive Rap1a localize to membrane protrusions. HeLa cells were transiently transfected with vectors encoding GFP-63E Rapla (63E) or GFP-17N Rap1a (17N) alone (left) or cotransfected with a HA-SWAP-70–encoding vector (right four panels). Transfected cells were suspended, plated on fibronectin for 1 h, fixed, and labeled with HA antibodies. Note that cells expressing 17N Rap1a do not spread, but when spreading is induced with SWAP-70, then 17N Rap1a is detected at the cell periphery. Arrowheads indicate localization of the GFP-Rap1a variants at the cell edge. (B) VAV2, but not COOL-1, requires Rap1 activity to localize to membrane protrusions. HeLa cells were transiently cotransfected with vectors encoding Myc-VAV2 or Myc-COOL-1 and GFP or GFP-Rap1GAP together with HA-SWAP-70. HA-SWAP-70 was cotransfected with the Rac GEFs and GFP vectors to allow Rap1-independent spreading. Transfected cells were treated as in A, labeled with Myc antibodies, and only well-spread cells were analyzed. Arrowheads indicate localization of the GEFs at the cell edge.
Mentions: As a second test for VAV2 localization, we examined the subcellular distribution of Rap1 and VAV2 in spreading cells using immunofluorescence (Fig. 7). Consistent with a recent paper (Bivona et al., 2004), a fraction of activated 63E Rap1a, but not dominant-negative 17N Rap1a, was found in membrane protrusions around the circumference of transiently transfected cells (Fig. 7 A, left). The absence of 17N Rap1a at the cell periphery conflicted with our pseudopodia fractionation studies and could be a secondary effect due to the lack of membrane protrusions around the circumference of cells in which Rap1 is inhibited. Accordingly, the Rac GEF SWAP-70, which induces cell spreading independently of Rap1, was expressed with 63E or 17N Rap1a. Consistent with the pseudopodia fractionation results (Fig. 6), both 63E Rap1a and 17N Rap1a were enriched in circumferential protrusions in SWAP-70–expressing cells (Fig. 7 A). These data suggest that both active and inactive Rap1 localize to Rac-dependent membrane protrusions. We examined the localization of the Rap1-dependent and -independent Rac GEFs VAV2 and COOL-1 in cells with normal or attenuated Rap1 activity. Overexpressed VAV2 and COOL-1 were both in peripheral membrane ruffles in cells with normal Rap1 activity. However, when Rap1 was inhibited with Rap1GAP, VAV2 but not COOL-1 was displaced from the cell periphery (unpublished data). Because the cells expressing VAV2 and Rap1GAP were poorly spread (Fig. 4), we again used SWAP-70 to induce Rap1-independent spreading (Fig. 7 B). Under these conditions, the cells were well-spread regardless of Rap1 activity, but VAV2 was displaced from circumferential membrane protrusions when Rap1 was inactivated. Together, these experiments suggest that active Rap1 relocalizes Rac GEFs that bind to Rap1, such as VAV2, to the periphery of spreading cells. In contrast, Rac GEFs that do not bind Rap1, such as COOL-1, accumulate in the periphery of spreading cells independently of Rap1 activity.

Bottom Line: Rap1 is necessary for the accumulation of VAV2 in membrane protrusions at the cell periphery.In addition, if VAV2 is artificially localized to the cell edge with the subcellular targeting domain of Rap1a, it increases cell spreading independently of Rap1.These results lead us to propose that Rap1 promotes cell spreading by localizing a subset of Rac GEFs to sites of active lamellipodia extension.

View Article: PubMed Central - PubMed

Affiliation: Fred Hutchison Cancer Research Center, Seattle, WA 98109, USA. barthur@fhcrc.org

ABSTRACT
The Ras-related GTPase Rap1 stimulates integrin-mediated adhesion and spreading in various mammalian cell types. Here, we demonstrate that Rap1 regulates cell spreading by localizing guanine nucleotide exchange factors (GEFs) that act via the Rho family GTPase Rac1. Rap1a activates Rac1 and requires Rac1 to enhance spreading, whereas Rac1 induces spreading independently of Rap1. Active Rap1a binds to a subset of Rac GEFs, including VAV2 and Tiam1 but not others such as SWAP-70 or COOL-1. Overexpressed VAV2 and Tiam1 specifically require Rap1 to promote spreading, even though Rac1 is activated independently of Rap1. Rap1 is necessary for the accumulation of VAV2 in membrane protrusions at the cell periphery. In addition, if VAV2 is artificially localized to the cell edge with the subcellular targeting domain of Rap1a, it increases cell spreading independently of Rap1. These results lead us to propose that Rap1 promotes cell spreading by localizing a subset of Rac GEFs to sites of active lamellipodia extension.

Show MeSH
Related in: MedlinePlus